Therapeutic potential and broader applicability of forced chromatin looping

 描述（由申请人提供）：增强子和启动子之间的长程相互作用在发育期间调节基因表达谱中起关键作用。基因座控制区（LCR）是一个强大的增强子，通过与胚胎、胎儿和成人珠蛋白基因启动子形成发育限制性长距离相互作用来激活β-珠蛋白基因。这些接触部分由称为Ldb 1的成环因子介导。该核因子占据LCR和球蛋白启动子两者，并且被认为通过自缔合促进成环相互作用。最近，我们的实验室已经表明，拴Ldb 1胎儿珠蛋白启动子通过人工锌指（ZF）蛋白触发启动子与LCR接触，并重新激活胎儿基因与高达85%的总珠蛋白合成培养的成人红系细胞。这是特别令人感兴趣的，因为高胎儿珠蛋白水平减轻血红蛋白病如镰状细胞病（SCD）的临床严重性。虽然通过这种方法达到的水平对于SCD患者是治疗性的，但是仍然不清楚这种策略是否可以应用于临床环境。为了实现这一目标，我们将在特异表达人类镰状血红蛋白的转基因小鼠中测试这种方法。这些小鼠含有编码胎儿和成人镰状β-珠蛋白的天然配置的人转基因。我们将用ZF-Ldb 1融合构建体或空载体从这些小鼠中扩增造血干细胞，并将转导的细胞移植到致死辐射的受体中。我们将比较ZF-Ldb 1处理的小鼠与空载体处理的小鼠的全血细胞计数、红细胞参数、器官病理学和存活率。此外，虽然强制染色质成环方法似乎是控制珠蛋白基因的稳健策略，但尚不清楚该策略是否可更广泛地适用。ChIP-seq研究表明，Ldb 1在整个基因组中占据了大量的位点。敲除实验表明，Ldb 1是广泛的红系基因的适当激活所必需的。然而，特定的长程相互作用Ldb 1可能介导的基因组范围内仍然不清楚。我们将以无偏见的方式为一组选择的Ldb 1结合的远端调控元件，在高分辨率与Capture-C方法，这是在我们的实验室建立了良好的远程相互作用的特点。为了深入了解Ldb 1全基因组介导的循环相互作用，我们将与Ali Mortazavi博士（UC Irvine）合作，使用配对末端标签测序（ChIA-PET）进行染色质相互作用分析。总之，所提出的实验旨在阐明染色质循环的基本机制，探索Ldb 1介导的循环治疗SCD的治疗潜力，并扩大该方法的范围到其他基因和疾病。

 DESCRIPTION (provided by applicant): Long-range interactions between enhancers and promoters play key roles in regulating gene expression profiles during development. The locus control region (LCR) is a powerful enhancer that activates ß-globin genes by forming developmentally restricted long-distance interactions with the embryonic, fetal, and adult globin gene promoters. These contacts are mediated in part by a looping factor termed Ldb1. This nuclear factor occupies both the LCR and globin promoters, and is thought to favor looping interactions by self- association. Recently, our laboratory has shown that tethering Ldb1 to the fetal globin promoter via artificial zinc finger (ZF) proteins triggers promoter contacts with the LCR, and re-activation of the fetal genes with up to 85% of total globin synthesis in cultured adult human erythroid cells. This is of particular interest as high fetal globin levels mitigate th clinical severity of hemoglobinopathies such as Sickle Cell Disease (SCD). While the levels achieved by this approach would be therapeutic for SCD patients, it is still unclear if this stratey can be applied in a clinical setting. Towards this goal, we will test this approach in transgenic mice that specifically express human sickle hemoglobin. These mice contain naturally configured human transgenes encoding the fetal and adult sickle ß-globin. We will transduce hematopoietic stem cells from these mice with either ZF-Ldb1 fusion constructs or empty vector and transplant the transduced cells into lethally irradiated recipients. We will compare complete blood counts, red blood cell parameters, organ pathologies, and survival rates of the ZF- Ldb1 treated mice to empty vector treated mice. Furthermore, while the forced chromatin looping approach appears to be a robust strategy to control globin genes, it is unclear if this strategy may be more broadly applicable. ChIP-seq studies have shown that Ldb1 occupies a substantial number of sites throughout the genome. Knockdown experiments have shown that Ldb1 is required for the proper activation of a wide range of erythroid genes. However, the specific long-range interactions Ldb1 may mediate genome-wide remain unclear. We will characterize long-range interactions in an unbiased manner for a select group of Ldb1-bound distal regulatory elements at high-resolution with the Capture-C method, which is well established in our laboratory. To gain insight into looping interactions mediated by Ldb1 genome-wide, we will use Chromatin Interaction Analysis using Paired-End Tag sequencing (ChIA-PET), in collaboration with Dr. Ali Mortazavi (UC Irvine). Together, the proposed experiments are designed to elucidate basic mechanisms of chromatin looping, to explore the therapeutic potential of Ldb1-mediated looping for the treatment of SCD, and broaden the scope of the approach to other genes and diseases.